RNA plays diverse biological roles, including in regulation and catalysis. The widespread distribution of self-cleaving RNAs (ribozymes) strongly suggests a biological significance. The folding and biochemical activity of most self-cleaving ribozymes are only dependent upon the presence of divalent metals and in a majority of ribozyme families there is no known regulatory mechanism of self-scission. In addition, the extent of structural flexibility in the domains peripheral to the ribozyme catalytic cores has neve been systematically characterized. The objective of this study is to uncover a new incidence of a self-cleaving ribozyme with regulated activity. The objective will be accomplished by: 1) using available software capable of searching for RNA structural motifs defined by the user, 2) creating new computational tools to assess the conservation of unique peripheral domains, 3) testing the activity of putative ribozymes for cleavage in vitro, and 4) determine if any cofactors are regulating ribozyme activity. The first step is to use structure-based searches to identify structural variants for two self-cleaving ribozyme families: hammerhead ribozyme (HHR) and hepatitis delta virus ribozyme (HDV). These computational searches use the unique secondary structure of each ribozyme family to guide a search through available genomic sequence databases for sequences capable of folding into the same motif. The second step builds bioinformatics tools to assess the conservation of predicted secondary structures in the ribozyme peripheral domains. The third step verifies the discovery of new ribozymes by testing for catalytic activity in vitro. Regulation of potential ribozymes will be inferred by taking into consideration conserved structural variations and consistent genomic locations of active ribozymes. The fourth step will use this information to predict candidate small molecules which may be involved in regulating ribozyme activity. The best candidates will be analyzed further for binding and the effect of binding on self-cleavage activity. The long-term objective of this study is to identify the biological roles of self-cleaving ribozymes in eukaryotic genomes. The study wil begin with two families of ribozyme (HHR and HDV) which are widespread throughout all kingdoms of life, including in the human genome, but for which the biological roles remain unclear. The implementation of the proposed research project will require the development of advanced computational tools and continued development of biochemical assays for RNA catalysis and ligand binding. Uncovering a mechanism regulating ribozyme activity may lead to the discovery of new regulatory pathways.